Chromosomal DNA replication is fundamental to the transmission of genetic information. In eukaryotes, each base pair of the genome is replicated once and only once per cell cycled. The initiation of replication, at origins, is the primary point of this cell cycle regulation. Little is understood about the regulation of initiation since chromosomal origins and the proteins that regulate initiation are just beginning to be identified. As a result of recent technical advances, it has become possible to assay for initiation of replication within yeast chromosomes. Consequently, origins of replication can be mapped to specific chromosomal regions. We have shown that coupling origin mapping experiments with site directed chromosomal mutagenesis allows direct identification of DNA sequence elements that are required for initiation of replication within the chromosome. We have mapped an origin of replication to the yeast HMR-E locus and identified two sequence elements that are required for initiation. One goal of this proposal is to extend this analysis to identify the additional elements that comprise this origin and thereby provide a detailed description of a eukaryotic chromosomal origin. The origin we study co-localizes with the HMR-E silencer, a DNA sequence element that represses transcription of the two nearby mating-type genes. Repression of transcription of these mating-type genes is an example of a position effect. Classically, position effects are revealed by repression of transcription of genes that either reside in or become translocated to inactive chromosomal regions. Other examples of position effects include X-chromosome inactivation in female mammals and position effect variegation in Drosophila. Current models propose that initiation of replication from the HMR-E silencer is required to establish the position effect. A second goal of this proposal is to critically test the idea that initiation of replication from a silencer-origin is required for the establishment of a position effect. Consistent with the idea that silencer-dependent initiation of replication is required for the position effect, we have recently shown that one protein that is required for the position effect is also required for initiation of chromosomal replication at the silencer-origin. The third goal of this proposal is to determine whether any of the several other proteins that are required for the position effect are also required for initiation of replication. Together, these experiments promise to begin to unravel the pathway that regulates initiation of replication, the mechanisms that impose position effects on chromosomes and the role that DNA replication may play in the regulation of transcription by position effects.